Tight junctions between cerebral endothelial cells normally prevent white cells from entering the central nervous system. Circulating polymorpho-nuclear leukocytes (PMNs) usually adhere only momentarily to the vessel wall. During an acute inflammatory process PMNs adhere to the endothelium and migrate into the brain along with plasma proteins, ions and water. The proposed experiments will attempt to define how the leukocytes cross the endothelial barrier and what factors are responsible for the increased permeability of this barrier. In order to better define the formation and disruption of the Blood- Brain Barrier several investigators have developed methods for the isolation and culture of brain microvessel endothelial cells. The cultured cells form confluent monolayers that contain Factor VIII/Von Willebrand antigen. We have previously demonstrated that the endothelial cells are bound together by tight junctions that restrict the para-cellular movement of horseradish peroxidase (HRP) and have very few cytoplasmic vesicles. These cultures thus provide a useful in vitro model for studies of cerebral microvessel function. We plan to use a new culture technique in order to study the adhesion and migration of leukocytes across the endothelial barrier. Endothelial cells isolated from bovine brain will be cultured in a chamber the floor of which consist of human amnion from which all epithelial cells have been previously removed. Human PMNs isolated by sedimentation in plasmagel will be placed on the surface of the monolayers in the upper chamber and a variety of potent neutrophil chemoattractants in the lower chamber. The cultures will be incubated for various periods of time and then fixed and processed for transmission electron microscopy. It is expected that in response to a gradient of chemoattractants many more PMNs will migrate across the endothelial cell layer than in the controls. Utilizing HRP as an electron dense tracer we will further investigate how the emigration of PMNs relates to changes in the permeability of the monolayers.